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Comprehensive identification, characterization, and expression analysis of the MORF gene family in Brassica napus

BMC Plant Biology volume 24, Article number: 475 (2024) Cite this article

Abstract

Background

RNA editing in chloroplast and mitochondrion transcripts of plants is an important type of post-transcriptional RNA modification in which members of the multiple organellar RNA editing factor gene family (MORF) play a crucial role. However, a systematic identification and characterization of MORF members in Brassica napus is still lacking.

Results

In this study, a total of 43 MORF genes were identified from the genome of the Brassica napus cultivar “Zhongshuang 11”. The Brassica napus MORF (BnMORF) family members were divided into three groups through phylogenetic analysis. BnMORF genes distributed on 14 chromosomes and expanded due to segmental duplication and whole genome duplication repetitions. The majority of BnMORF proteins were predicted to be localized to mitochondria and chloroplasts. The promoter cis-regulatory element analysis, spatial-temporal expression profiling, and co-expression network of BnMORF genes indicated the involvement of BnMORF genes in stress and phytohormone responses, as well as growth and development.

Conclusion

This study provides a comprehensive analysis of BnMORF genes and lays a foundation for further exploring their physiological functions in Brassica napus.

Peer Review reports

Background

As a post-transcriptional RNA metabolic process, RNA editing generally modifies the genetic information present in RNA molecules through nucleotide insertion, deletion, or conversion [1]. In plants, RNA editing occurs mainly in the two organelles with resident genomes: chloroplast and mitochondrion, involving the cytidine to uridine (C-to-U) modification (rarely U-to-C) within coding or noncoding regions of RNAs [2, 3]. There are about 20 to 40 and 400 to 600 conserved C-to-U editing sites in the chloroplast and mitochondrion genome of most flowering plants, respectively [4]. In plants, RNA editing plays important roles in organelle biogenesis, organelle signaling, and adaptation to environmental stresses. Previous studies showed that many mutant plants with impaired site-specific RNA editing exhibited strong deleterious phenotypes, even lethal ones [2, 5,6,7,8].

In plants, the RNA editing process is conducted by molecular machinery called the RNA editosome, which is formed by various nuclear-encoded RNA editing factors. These factors include pentatricopeptide repeat proteins (PPR), multiple organellar RNA editing factor (MORF) proteins/RNA editing factor interacting proteins (RIPs) [3, 9, 10], chloroplast ribonucleoproteins (cpRNPs), organelle RNA recognition motif proteins (ORRM) [11, 12], organelle zinc-finger proteins (OZ) [13], and protoporphyrinogen oxidase 1 (PPO1) [14]. In RNA editosomes, PPR proteins bind to RNA molecules and recognize RNA editing sites. The PPR-RNA complex is structured into editosomes together with many non-PPR protein factors. These non-PPR proteins, including MORF and ORRM proteins, might play as connectors of site-specific PPR proteins with other RNA editing factors or editing efficiency regulators. There are ten MORF family members in Arabidopsis thaliana. MORF2 and MORF9 are chloroplast-localized, MORF8 is dual-localized in the mitochondrion and chloroplast, and the remaining MORF proteins are mitochondrion-localized. Five members of the MORF family act as key RNA editing factors in mitochondria and/or chloroplasts, whereas the rest of the MORFs have less or no impact on RNA editing [9, 10]. It was reported that disrupting the MORF1, MORF3, and MORF8 genes decreased the editing efficiency at 19%, 26%, and 72% of mitochondrial RNA editing sites, respectively. The loss of function of MORF2 or MORF9 abolished the RNA editing efficiency at almost all chloroplast editing sites [10, 15].

Members of the MORF family have also been identified in several different plant species, including Populus trichocarpa with nine [5], Oryza sativa with seven [16], Zea mays with seven [17], and Nicotiana benthamiana with eight members [18]. MORF members contain a centrally conserved MORF domain composed of a core of six anti-parallel β-sheets flanked on one side by three α-helices and several loops on the other side [19, 20]. MORF proteins can form homodimers and heterodimers and selectively interact with other RNA editing factors, such as site-specific PPR proteins, ORRM proteins, and PPO1 [3]. Furthermore, MORF members may be essential to the growth, development, and stress responses of plants. In Arabidopsis, leaves of morf2 and morf9 mutants showed decreased chlorophyll content, and morf8 mutant plants exhibited a dwarf phenotype [9, 21]. In rice, mutant plants of WSP1 encoding a protein from the MORF family had reduced chlorophyll content and emerged with a white immature panicle phenotype [22], and the expression of rice MORF genes was affected by cold and salt treatments [16]. In tobacco, NbMORF8 has been demonstrated to negatively regulate the immunity of plants to pathogens [18]. The poplar PtrMORF genes have also been found to respond to drought stress [5].

The key oil crop, Brassica napus, is grown extensively all over the world and is one of the most significant sources of sauces, vegetables, and industrial oil [23]. Brassica napus (AnAnCnCn, 2n = 38) developed naturally from the hybridization of two diploid species, Brassica rapa (B. rapa; AnAn, n = 10) and Brassica oleracea (B. oleracea; CnCn, n = 9) [24, 25]. However, the genome-wide identification and characterization of the Brassica napus MORF (BnMORF) gene family is still lacking. In this study, members of the BnMORF gene family were identified and analyzed, including their gene structures, chromosomal localizations, and evolutionary patterns. The expression profile of BnMORF genes in various tissues and under different phytohormone and stress treatments was explored. The cis-regulatory elements and transcription factor binding sites in promoters of BnMORF genes were also identified. This study will help us better understand the MORF gene family and establish a basis for future exploration and functional validation of MORF genes in Brassica napus.

Results

Identification and classification of the Brassica napus MORF (BnMORF) gene family members

To obtain all the candidate Brassica napus MORF gene family members, we performed a BLASTP homology search against the Brassica napus cultivar Zhongshuang 11 (ZS11) genome using Arabidopsis MORF protein sequences as queries. Subsequently, by confirming the presence of the MORF domain (IPR039206) in candidate BnMORF members and removing the redundant gene forms simultaneously, 43 BnMORF genes were genome-widely identified from the ZS11 genome (Table 1). All the identified BnMORF genes were then given new names according to their chromosome locations, and their gene IDs from different versions of genome annotation were also identified (Table 1). As shown in Table 2, the BnMORF proteins have a length ranging from 116 to 910 amino acids, and the average value was 297.44 amino acids. The isoelectric point (pI) of BnMORF proteins varies from 5.09 to 9.75, with an average value of 8.23. Their molecular weight (MW) varies from 13.56 to 98.84 kDa, with an average value of 32.91 kDa. Prediction of subcellular localization revealed that ten BnMORF proteins were identified in chloroplasts, while 27 BnMORF proteins are localized to mitochondria, and the remaining six proteins are localized elsewhere.

Table 1 The list of identified BnMORF genes in Brassica napus by this study
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Table 2 Protein features of the 43 BnMORF family members identified in Brassica napus cultivar ZS11
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Phylogenetic and sequence structural features of BnMORF family members

To investigate the classification and evolutionary relationship of the MORF members, an unrooted phylogenetic tree using the neighbor-joining method was constructed from 67 MORF protein sequences collected from maize (Zea mays), Arabidopsis thaliana, Brassica napus, and rice (Oryza sativa) (Additional file 1: Fig. S1). Based on the phylogenetic tree, all the MORF proteins can be subdivided into three groups, named I to III. The size of the three groups varies. Group II is the largest group, containing 23 MORF members (Additional file 1: Fig. S1). After the lineage separation, species-specific gene duplications occurred, resulting in the inclusion of multiple MORF genes per species. The phylogenetic tree only containing the 43 BnMORF members showed that BnMORF members were also divided into three similar groups, according to homology relationships (Fig. 1). BnMORF members are equivalent in groups I and II, with 15 members, while group III has 13.

Fig. 1
figure 1

Neighbor-joining phylogenetic tree of BnMORF family members. The topology was assessed with a bootstrap analysis with 1000 replicates. All BnMORF members are divided into groups I–III. The bootstrap values are displayed, and various groups are denoted by distinct background colors. The length of the branches represents evolutionary distances, and the scale bar indicates 0.1 substitutions each point

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Exon-intron structural changes in genes and motif composition in proteins both play vital roles in gene/protein functional differentiation. Motif composition and the intron/exon structure of each BnMORF member were analyzed (Fig. 2). The BnMORF genes contained several exons ranging from three to nine. BnMORF4 contained only three exons, while BnMORF13 and BnMORF34 contained nine exons (Fig. 2a). Most BnMORF genes within the same phylogenetic tree clan or in the same group presented similar exon-intron distribution patterns. We annotated conserved motifs in BnMORF proteins using the MEME server, and one to ten conserved motifs were found in BnMORF family members (Fig. 2b and c). All BnMORF proteins except BnMORF37 and BnMORF39 contain the highly conserved motif 1, which consists of 34 amino acids. Except for BnMORF4, BnMORF24, and BnMORF28, all the other BnMORF proteins have the 38-amino acid conserved motif 2 (Fig. 2b and c). Some motifs specifically exist in BnMORF members from the same phylogenetic tree clan, and they may link to unique biological functions.

Fig. 2
figure 2

Comparison of conserved protein motifs and gene structures of BnMORF members. (A) Schematic exon/intron structures of BnMORF genes. The exons are indicated by yellow rectangles, and the introns are represented by black lines. The UTR regions are indicated by green rectangles. (B) Conserved motifs in BnMORF proteins. Ten motifs are indicated by different colored rectangles. (C) The sequence logos of the ten conserved motifs. The x-axis represents the conserved sequence of each domain. The y-axis represents a measure of relative entropy, and the height of each letter indicates the rate of conservation of each amino acid across all proteins

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Chromosomal locations and collinearity analysis of BnMORF genes

The chromosomal localization analysis revealed that 43 BnMORF genes were scattered irregularly across 14 chromosomes. The number of genes on each chromosome has no bearing on chromosome size (Fig. 3). No BnMORF genes are located on chromosomes A02, A09, A10, C02, and C09. There are 20 BnMORF genes located on the A subgenome, while the rest of the BnMORF genes are located on the C subgenome. Chromosomes A05 and C03 both contain the largest number of five BnMORF genes, while chromosomes A08 and C08 both contain only one BnMORF gene (Fig. 3). To investigate the evolution of BnMORF genes, the synteny between Brassica napus and Arabidopsis at the whole genome level was analyzed. Between the two genomes, 40 collinear MORF gene pairs were identified (Fig. 4, Additional file 2: Table S1). All Arabidopsis MORF (AtMORF) genes have multiple syntenic BnMORF genes. For example, both AtMORF1, AtMORF4, and AtMORF8 have six collinear BnMORFs. However, AtMORF5 has only two synthetic BnMORFs (Additional file 1: Table S1). We also investigated the duplication events of BnMORF family genes by BLASTP and MCScanX. We discovered that BnMORF genes were mainly derived from whole genome duplication and segmental duplication events (Additional file 3: Table S2). In addition, intra-species collinearity analysis of 43 BnMORF genes revealed 76 collinear gene pairs (Fig. 5, Additional file 4: Table S3). These results indicated that segmental duplication and whole genome duplication appeared to play a major role in the expansion of the BnMORF gene family.

Fig. 3
figure 3

The chromosomal distribution of BnMORF genes. The chromosome numbers are indicated at the top of every chromosome, and each chromosome distance is displayed in megabytes (Mb) at the bottom. The BnMORF gene names are highlighted in black

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Fig. 4
figure 4

The synteny analysis of MORF family members between Brassica napus and Arabidopsis. The collinear blocks generated by the Brassica napus and Arabidopsis thaliana genomes are indicated with gray lines in the background, whereas syntenic MORF gene pairs are indicated with red lines

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Fig. 5
figure 5

Intra-species collinearity analysis of BnMORF family members. BnMORF genes are mapped to 14 Brassica napus chromosomes in a circle, and segmental duplications are mapped to their respective locations. The red lines represent the homologous relationships among BnMORF family members, and the gray lines represent all background collinear pairs

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The cis-regulatory elements and transcription factor binding sites analysis in promoters of BnMORF genes

Cis-regulatory elements and transcription factor binding sites are crucial in modulating gene expression, and promoters of genes with related functions may contain similar regulatory elements. We also carried out the cis-regulatory elements and transcription factor binding sites analysis in promoters of BnMORF genes. As displayed in Fig. 6, the identified cis-regulatory elements in the promoters of BnMORF genes can be classified into four main categories. The first category is for light reactions. The promoters of all BnMORF genes contained several light-responsive elements, the majority of which were AE-box elements. The elements of development and growth, including circadian regulation, endosperm expression, and meristem expression, were covered in category two. Abscisic acid, auxin, gibberellin, methyl jasmonate, and salicylic acid were the members of category three. At least one cis-regulatory element involved in phytohormone responsiveness classification was found in all BnMORF genes. Further investigation revealed that 37 genes carried methyl jasmonate responsive elements, 35 carried abscisic acid responsive elements, 21 carried auxin responsive elements, 14 carried salicylic acid responsive elements, and 27 genes carried gibberellin responsive elements (Additional file 5: Table S4). Category four is associated with abiotic stresses such as drought inducibility, low-temperature, and anaerobic induction. There were cis-regulatory elements for anaerobic induction in 38 genes, for low-temperature responsiveness in 21 genes, and for drought induction in 20 genes, indicating that BnMORF genes may be factors responding to abiotic stresses. Binding sites of 25 TF families, including Trihelix, ERF, GRAS, C2H2, MYB_related, BBR-BPC, B3, Dof, Nin-like, LBD, GRF, G2-like, bHLH, bZIP, GATA, NAC, EIL, MIKC_MADS, MYB, E2F/DP, AP2, CPP, SBP, SRS, ZF-HD, and HD-ZIP, were identified in promoters of BnMORF genes (Fig. 7). The ERF family covers 16 BnMORF members, while the SRS and ZF-HD families cover only one family member, respectively (Fig. 7).

Fig. 6
figure 6

Predicted cis-regulatory elements in promoters of BnMORF genes. On the right side, the names of each cis-regulatory element are displayed. Different colored boxes in the gray rectangle represent different cis- regulatory element. The scale can be utilized for estimating each element’s relative position. The direction of promoter sequence is from 5’ to 3’

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Fig. 7
figure 7

Predicted transcription factor binding sites in BnMORF promoters. Boxes of different colors represent the binding sites of different transcription factors. The names of various transcription factor families are shown on the right

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The expression pattern of BnMORF genes at different developmental stages

The study of a gene’s expression pattern in various tissues or organs will help to unravel its biological function. The BnIR database was utilized for obtaining the expression data of BnMORF genes in different tissues or organs (root, stem, cotyledon, vegetative rosette, leaf, bud, flower, silique, and seed) throughout various developmental stages. Excluding for 11 genes that showed no detectable expression levels (TPM < 0.5) in any of the samples tested, the remaining 32 genes had obvious preferential expression patterns (Additional file 6: Table S5). The 32 BnMORF genes are clustered into three groups based on their expression patterns (Fig. 8). Nearly all the BnMORF genes have relative high expression in the bud and silique of early development stages. Furthermore, in group I, all 10 genes preferentially express in the cotyledon, vegetative rosette, and young silique, except that BnMORF14 shows obvious leaf and bud-specific expression. In group II, most members also show relative high expression in root and seed, and BnMORF16 and BnMORF26 display specific high expression in root and young seed. In group III, most genes have relative high expression in seed, except that BnMORF37 and BnMORF38 present stem-specific expression (Fig. 8).

Fig. 8
figure 8

The expression pattern of BnMORF genes in different tissues or organs of Brassica napus cultivar ZS11 at different developmental stages. The expression profile of the 32 BnMORF genes are displayed in a hierarchical cluster. At the bottom of each column, tissues or organs from various developmental phases of Brassica napus cultivar ZS11 employed for expression profiling are noted. On the left is a cluster dendrogram. The color key represents the Z-score values transformed from the expression values. Ro: root; LSP: lower stem peel; MSP: middle stem peel; USP: upper stem peel; Le: leaf; Co: cotyledon; VR: vegetative rosette; Bd: bud; Si: silique; SW: silique wall; Sd: seed; d indicates day

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The expression profile of BnMORF genes in response to different stress and phytohormone treatments

We also investigated the modulation of BnMORF gene expression in response to various abiotic stress and phytohormone treatments (Additional file 7: Table S6). The expression of BnMORF genes of groups I and III (except BnMORF36 and BnMORF37) classified by different developmental stages (Fig. 8) was preferential induced in leaves other than in roots, while the accumulation of transcripts of genes of group II was more abundant in roots (Fig. 9a). We discovered that the transcript levels in leaves of most BnMORF genes except BnMORF37 were significantly induced (more than 2-fold) after being exposed to heat for 12 h, and the expression of BnMORF genes belonging to group II (except BnMORF42) in roots was more responsive to heat stress in roots. In addition, the expression of BnMORF genes in group I (except BnMORF14 and BnMORF34) was significantly increased in the leaves with 3 h of freezing treatment. The expression in the roots of BnMORF32, BnMORF7, BnMORF26, and BnMORF29, which belonged to group II, was also strongly induced. Notably, BnMORF37, BnMORF19, BnMORF34, and BnMORF14 had low expression levels in leaves under normal conditions, but were considerably up-regulated in leaves under salt stress, indicating potential involvement in leaves in response to salt stress (Fig. 9a). These expression patterns suggested that BnMORF members may have significant response functions to abiotic stress, especially heat and cold stress. We also found that gibberellin (GA) and abscisic acid (ABA) treatment all decreased the expression of group I genes in leaves and the expression levels of group II genes in roots (Fig. 9b). Surprisingly, the mRNA accumulation in group I BnMORF genes (except for BnMORF14) in leaves fluctuated obviously, exhibiting increased expression after IAA treatment. The expression of group II BnMORF genes (except BnMORF22 and BnMORF26) in roots increased significantly after three hours of IAA treatment. We subsequently selected 10 BnMORF genes exhibiting significant expression level changes in response to heat and IAA treatment and used quantitative reverse transcription PCR (RT-qPCR) to validate their expression patterns. The results showed that the expression levels of these 10 BnMORF genes were significantly up-regulated in leaves or roots after heat or IAA treatments (Fig. 9c and d), which was consistent with the above expression profile obtained from the BnIR transcriptome database.

Fig. 9
figure 9

The expression profile of BnMORF genes under different stress and phytohormone treatments. (A) The expression pattern of BnMORF genes in leaf and root under salt, drought, freezing, and heat treatments at different time points. (B) The expression pattern of BnMORF genes in leaf and root treated phytohormone with IAA, GA, ABA, and BL at different time points. Z-score normalization was used to process the expression data. The color scale represents relative expression levels from low (green) to high (red). h indicates hour. (C) The expression level of BnMORF genes revealed by RT-qPCR in leaves and roots under heat treatment. Data are mean ± SEM from two biological replicates, and asterisks denote significant differences using a two-tailed Student’s t test (*p-value < 0.05, **p-value < 0.01, ***p-value < 0.001). (D) The expression level of BnMORF genes revealed by RT-qPCR in leaves and roots under IAA treatment. Data are mean ± SEM from two biological replicates, and asterisks denote significant differences using a two-tailed Student’s t test (*p-value < 0.05, **p-value < 0.01, ***p-value < 0.001)

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A co-expression network of BnMORF genes responding to different stresses was also constructed (Fig. 10). BnMORF43 was the top hub gene in the network with the highest degree and betweenness centrality, with 11 BnMORF genes directly linked to it. Interestingly, the co-expression genes such as BnMORF43, BnMORF30, BnMORF12, BnMORF33, and BnMORF20 had the same expression pattern across the tissues and in response to different abiotic stress and hormone treatments (Figs. 8 and 9). These results indicate that these BnMORF genes may be co-regulated, functionally linked, or involved in the same signaling pathway or physiological activity.

Fig. 10
figure 10

The co-expression network of BnMORF genes in response to different stresses. Each gene’s hue is determined by its degree and betweenness centrality. The red genes indicate strongly connected nodes, whereas the yellow genes indicate less connected nodes. Positive and negative correlations are represented by the red and blue lines between genes, respectively

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Discussion

In general, RNA editing in plants is considered a DNA mutation correction process at the RNA level, restoring conserved amino acids to guarantee that proteins continue to function normally [15]. Plant MORF proteins are multifunctional proteins that interact with other RNA editing factors and play an essential role in chloroplast gene editing [7]. Moreover, recent studies demonstrated that MORF genes were important for growth of plants and responses to stressful conditions, including survival of seeds in rice [16], pathogen stress in tobacco and kiwifruit [18, 26], and drought-related stress in poplar [5]. However, no research on the MORF gene family in Brassica napus has been documented. We identified 43 BnMORF family members in Brassica napus using the recently published ZS11 genome. Subcellular localization prediction revealed that 63% of BnMORF members were localized to mitochondria and 23% to chloroplasts, which was consistent with the fact that most MORF proteins in Arabidopsis are mitochondrion localization and a few are localized to chloroplasts [10]. Subcellular localization prediction revealed that four homologs of AtMORF2 in Brassica napus, BnMORF8, BnMORF12, BnMORF30, and BnMORF31, are localized to chloroplasts. Moreover, the corresponding genes of these four BnMORF proteins were highly expressed in green tissues such as the vegetative rosette and siliques. The gene structure suggested that most BnMORF members within the same phylogenetic tree clan presented similar exon-intron distribution patterns as well as motif compositions. We hypothesized that the differences in physical and chemical properties of BnMORF proteins might be caused by the diversity of gene structure and motif compositions.

Gene replication plays an essential role in the evolution of species, mainly through whole genome replication, segmental and tandem replications [27]. Brassica napus developed naturally from the hybridization of two diploid species, and its diploid parents also experienced Arabidopsis-based polyploidization events [24]. In theory, after genome-wide polyploidy, Brassica napus should have six homologous genes for each Arabidopsis gene. However, only 43 BnMORF family members were identified in this study, suggesting that about 20% of MORF genes had been lost during evolution. Although AtMORF genes have multiple direct homologs in Brassica napus, only AtMORF1, AtMORF4, and AtMORF8 had six homologous genes, according to the interspecific collinear study of Arabidopsis and Brassica napus. The results showed that other AtMORF homologous genes experienced complicated events such as gene expansion and loss during the evolution of Brassica napus. The collinearity analysis also showed that most BnMORF family members were generated by whole genome duplication or segmental duplication. The MORF genes in the Brassica napus genome were expanded to some extent even if gene loss happened during the evolution of Brassica napus, indicating that gene duplication played a significant role in the development of Brassica napus.

It was hypothesized that tissue-specific expression of MORF genes would determine their functional divergence. Whole growth period expression profiling analysis of BnMORF genes revealed that most BnMORF genes were universally expressed in green tissues such as buds and young siliques. Some genes were expressed in stems and roots specifically, indicating the expanded function of BnMORF genes. These results indicated that BnMORF genes may function closely in various tissues and organs. In addition, we found that BnMORF genes that are closely related in evolution have similar expression patterns. An increasing number of studies have shown that RNA editing events in chloroplast and mitochondrial genes play an important role in response to a variety of environmental stresses. Low temperatures affect the processing of wheat mitochondrial cox2 transcripts and reduce the efficiency of RNA editing [28]. When maize was exposed to 37℃, the editing efficiency of rps14 and rpl20 transcripts decreased by 70% [29]. In different strains of rice, more than half of the 90 editing sites in transcripts of six mitochondrial genes were sensitive to oxidative stress treatment, and the editing efficiency of these sites were modified by oxidative stress [30]. We discovered that promoters of BnMORF genes possessed a variety of cis-regulatory elements and transcription factor binding sites associated with abiotic stress and phytohormone responses. Moreover, the expression of many BnMORF genes was regulated by different abiotic stress and hormone treatments. This implied that BnMORF genes might also been engaged in the development and growth of rapeseed by responding distinct abiotic stress and phytohormone regulatory pathways through their roles in chloroplast and mitochondrial RNA editing.

Conclusions

In this study, 43 BnMORF genes were identified from the latest annotated version of the Brassica napus cultivar “Zhongshuang 11” genome. Phylogenetic analysis divided BnMORF gene family members into three groups, and this classification was further supported by similar conserved motif compositions and exon-intron distributions. The collinearity analysis showed that segmental and whole genome duplication had a significant impact on the expansion of the BnMORF gene family. A number of cis-regulatory elements and transcription factor binding sites related to abiotic stress and hormone responses were also identified in promoters of BnMORF genes. Transcriptome data also revealed that BnMORF genes might participate in growth and development, as well as abiotic stress and hormone responses. Taken together, these findings provide a foundation for further exploring the functions of MORF genes in Brassica napus.

Methods

Identification of MORF family members in Brassica napus

Taking the Brassica napus cultivar “Zhongshuang 11” (ZS11) ZS11.v10 genome as a reference, the candidate BnMORF family members were identified by BLASTP search in the Brassica napus genome database (http://yanglab.hzau.edu.cn/BnIR/BLAST/) [31] using MORF protein sequences from Arabidopsis as queries and the e-value ≤ 5 as a threshold. We retrieved the protein sequences and gene model annotation files from BRAD (http://www.brassicadb.cn/). InterProScan (http://www.ebi.ac.uk/interpro/) was further used to confirm the existence of the MORF domain (IPR039206) in candidate proteins. Sequences without MORF domains were removed. According to the chromosomal locations of corresponding genes, the remaining BnMORF members were named sequentially. The molecular weight and isoelectric point of BnMORF proteins were calculated using the protparam (https://web.expasy.org/protparam/) [32] In addition, subcellular localization and chloroplast/mitochondrion transit peptides of BnMORF proteins were predicted by TargetP2.0 (https://services.healthtech.dtu.dk/services/TargetP-2.0/) and PredSL (http://bioinformatics.biol.uoa.gr/).

Phylogenetic tree construction

In addition to Brassica napus, we also used MORF members from three more species (Arabidopsis thaliana, Oryza sativa, and Zea mays) for phylogenetic analysis to explore the function and molecular evolution of BnMORF members. The muscle method was utilized to align the MORF protein sequences, and the tree of phylogenetic relationships was built in MEGA7 software using the neighbor-joining method with the Possion model, 1000 bootstrap values, and pairwise deletion [33]. Besides, the phylogenetic tree among MORF members in Brassica napus was also constructed using the above method. The phylogenetic trees were further embellished using ITOL (http://itol.embl.de/).

Conserved motif and gene structure analysis

The predicted introns and exons in BnMORF genes were extracted from the GFF3 file of Brassica napus ZS11.v10, and their intron and exon structures were visualized by Gene Structure View of TBtools software. We explored the conserved motifs in BnMORF proteins utilizing the MEME motif suite (https://memesuite.org/meme/index.html/) [34] and used the TBtools to visualize them [35].

Chromosomal location and collinearity survey of BnMORF genes

BnMORF genes were mapped to the Brassica napus genome using MG2C (http://mg2c.iask.in/mg2c_v2.1/). Chromosome length, gene location, and length information from the GFF3 annotation file were extracted by TBtools. The synteny links between orthologous Arabidopsis and Brassica napus MORF genes were determined using the dual-synteny-plotter function of TBtools. With default options, the Multiple Collinearity Scan Toolkit (MCScanX) was used to evaluate the gene duplications of the BnMORF family members, and Circos (http://circos.ca/) was used to display the collinearity information [36, 37].

Spatial-temporal and abiotic stress expression profile analysis of BnMORF genes

The expression data of BnMORF genes was obtained from the expression profile (ZS11 library) of BnIR. The genes with TPM (transcripts per million) < 0.5 were excluded from further analysis. Z-score normalization was used to process the expression data and heatmaps were generated by TBtools. The co-expression analysis was performed using the Pearson correlation coefficient and visualized by Cytoscape [38, 39]. We obtained 14 RNA-seq datasets on BnMORF gene responses to different stresses from the BnIR expression profile (ZS11 library) for co-expression analysis.

Promoter analysis and transcription factor binding site prediction

Genomic DNA sequences up to 2000 bp upstream of the start codon of each BnMORF gene were extracted from the Brassica napus multi-omics information resource database (BnIR, https://yanglab.hzau.edu.cn/), and then PlantCare software (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) was used to predict cis-regulatory elements in the promoter region of BnMORF genes. In addition, we use the PlantTFDB website (http://planttfdb.cbi.pku.edu.cn/) to predict transcription factor binding sites with a setting of p-value ≤ 1e− 6. TBtools software was used to visualize the number and distribution of cis-regulatory elements and transcription factor binding sites.

RT-qPCR analysis of BnMORF genes under heat and IAA treatments

The expression level of BnMORF genes under heat and IAA treatments was examined by RT-qPCR. Brassica napus ZS11 plants were grown on 1/2 LS solid medium under long-day conditions with 100 µmol·m− 2·s− 1 light intensity at 22 °C for 14 days. For heat treatment, seedlings were placed at 38 °C for 3 h, followed by recovery at 22 °C for 12 h. For IAA treatment, 14-day-old seedlings were treated with 10 µM IAA for 3 h. Following the treatment, root and leaf samples were collected, respectively. Root and leaf samples collected from untreated plants were served as controls [31]. Total RNA was isolated using the RNAprep Pure Plant Kit (DP432, Tiangen, China). The first-strand cDNA was synthesized using the HiScript III 1st Strand cDNA Synthesis Kit (+ gDNA wiper) (R312-02, Vazyme, China) with the addition of Oligo (dT)20VN and random hexamer primers. The primers used in RT-qPCR analysis were listed in Additional file 8: Table S7. RT-qPCR was performed using the Bio-rad CFX Connect Real-Time PCR Detection System and the ChamQ Blue Universal SYBR qPCR Master Mix (Q312-02, Vazyme, China). PP2A (Protein phosphatase 2 A subunit A3), which showed suitability across multiple conditions, was used as the reference gene [40]. The relative expression level of each gene was calculated by the 2−ΔΔCt method [41].

Data availability

All the data supporting the results of this article is included in the article and additional files.

Abbreviations

MORF:

Multiple organellar RNA editing factor

BnMORF:

Brassica napus MORF

RIP:

RNA editing factor interacting protein

ZS11:

Zhongshuang 11

pI:

Isoelectric point

aa:

Amino acids

TF:

Transcription factor

TPM:

Transcripts per million

RNA-seq:

RNA sequencing

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Acknowledgements

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Funding

This work is supported by grants from the National Natural Science Foundation of China (32170556). X.Z. is supported by the Excellent Young Scientists Fund Program (Overseas) from National Natural Science Foundation of China and the Hundred-Talent Program of Zhejiang University.

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  1. Jiani Xing and Yayi Zhang contributed equally to this work.

Authors and Affiliations

  1. Key Laboratory of Nuclear Agricultural Sciences of Ministry of Agriculture and Rural Affairs, Key Laboratory of Nuclear Agricultural Sciences of Zhejiang Province, Institute of Nuclear Agricultural Sciences, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, 310058, China

    Jiani Xing, Yayi Zhang, Wenjian Song, Nadia Ahmed Ali, Kexing Su, Xingxing Sun, Yujia Sun, Yizhou Jiang & Xiaobo Zhao

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J.X., Y.Z. and X.Z. conceived and designed the study. J.X., Y.Z., W.S., N.A.A., K.S., X. S., Y.S. and Y.J. performed the research and analyzed the data. J.X., Y.Z., and X.Z. wrote the manuscript. X.Z. supervised the research. All authors have read and approved the manuscript.

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Xing, J., Zhang, Y., Song, W. et al. Comprehensive identification, characterization, and expression analysis of the MORF gene family in Brassica napus. BMC Plant Biol 24, 475 (2024). https://doi.org/10.1186/s12870-024-05177-3

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BMC Plant Biology

ISSN: 1471-2229

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